<Emphasis Type="Italic">Volvox</Emphasis> (Chlorophyta,Volvocales) as a model organism in developmental biology

Model systems based on two or more related species with different types of development are finding increasing use in current comparative embryology. Green algae of the genus Volvox offer an interesting opportunity to study sex pheromones, morphogenesis as well as the formation of a somatic cell line undergoing terminal differentiation, senescence, and death as well as a line of reproductive cells, which at first grow and then undergo a series of consecutive divisions that give rise to new organisms. However, almost all studies of the recent years were conducted on a single species, Volvox carteri f. nagariensis. The goal of this publication was to advertise the cosmopolitan alga V. aureus as a model species in developmental biology. Published data on V. aureus are briefly reviewed in comparison with the development of V. carteri and outlooks of further studies are specified. In particular, the expediency of collecting new V. aureus strains from nature to study their development in clonal culture is outlined.     

The sexual inducer of Volvox carteri: purification, chemical characterization and identification of its gene

The sexual inducer of Volvox carteri f. nagariensis is a glycoprotein and one of the most potent biological effector molecules known. It is synthesized by sperm cells and converts asexually growing males and females to the sexual pathway. Until now, large-scale production of the inducer was made impossible by an inherent biological `switch' mechanism, the spontaneous self-induction of asexually growing males. Here we describe a method overcoming this problem for the first time. Large-scale production and purification allowed a detailed chemical characterization of the inducer with respect to partial amino acid sequences and sugar composition. Chemically synthesized oligodeoxynucleotides corresponding to derived amino acid sequences were used to screen a genomic gene bank of V. carteri HK 10. A positive clone (Ind-28) was shown to encode the inducer gene by subcloning and sequencing.     

A Preliminary Genetic Investigation of VOLVOX CARTERI

A preliminary genetic analysis of a number of genetic variants of Volvox carteri f. nagariensis is presented. Techniques are outlined for mutagenesis of Volvox, isolation of mutants and routine genetic analysis. All of the mutants show simple Mendelian segregation patterns and have been tentatively placed in 14 linkage groups.     

Mitosis and cytokinesis in androgonidia ofVolvox carteri f.weismannia

Summary Reproductive cells (androgonidia) ofVolvox carteri f.weismannia divide to form packets of 64 or 128 sperm cells. The androgonidium morphology, stages of mitosis, and cytokinesis were examined by electron microscopy. The biflagellate androgonidium loses its flagella before mitosis but the flagellar bases at the anterior end of the cell are retained. Two additional basal bodies are formed and the nucleus migrates from its central position to the area of the basal bodies before mitosis begins. A five-layered kinetochore is present on the chromosomes and remnant nucleolar material persists during mitosis. A furrow at the chloroplast end of the cell and the formation of phycoplast microtubules and vesicles signal the beginning of cytokinesis at early telophase. The cells maintain cytoplasmic connections until after the packet of sperm cells completes its development.     

Mutants of Volvox carteri affecting nitrogen assimilation

Summary Three genes of Volvox carteri f. nagariensis have been identified which affect nitrogen assimilation. Mutants in two unlinked genes, nitA and nitC, were isolated as chlorate resistant and they exhibit no measureable nitrate reductase activity. The mutant in the nitB gene which is linked to nitA has slightly reduced levels of nitrate reductase activity and grows poorly on the nitrate concentration in standard medium but grows normally if the level of nitrate is increased. All of the mutants utilize ammonia, urea or nitrite for growth.Deseased     

FEULGEN MICROSPECTROPHOTOMETRIC STUDIES OF PANDORINA MORUM AND OTHER VOLVOCALES (CHLOROPHYCEAE)12

The nuclear DNA content during normal vegetative growth and division has been examined in three species of Volvocales, Chlamydomonas reinhardtii Dangeard, Pandorina morum Bory, and Volvox carteri f. nagariensis Iyengar. The results are consistent with the nuclear cycle reported in the literature for Eudorina. Nuclear DNA content does not increase during the prolonged cell growth phase. At the time of colony formation, nuclear DNA doubles, the nucleus divides, and this alternation continues until the final 2ⁿ complement of progeny nuclei is formed. The 4- and 8-nucleate stages of dividing gonidia of V. carteri have a nuclear DNA content in the same range as the somatic cells; they are not polyploid or polytene. Four normal clones of Pandorina, having 2, 5 or 12 chromosomes, all had similar amounts of DNA per nucleus, suggesting that the species has a nuclear genome of fairly constant size rather than consisting of many strains representing a polyploid series. One unique clone, a hybrid with double the chromosome number of either its parents, had twice as much DNA as the normal clones. The Feulgen spectrophotometric method is sufficiently sensitive to detect 2-fold differences in DNA content at the level of 2 × 10^?13 g of DNA /nucleus, and its use avoids the complications associated with the presence of organelle DNA.     

SOMATIC CELL FLAGELLAR APPARATUSES IN TWO SPECIES OF VOLVOX (CHLOROPHYCEAE)1

The somatic cell flagellar apparatuses of Volvox carteri f. weismannia (Powers) Iyengar and V. rousseletii G. S. West have parallel or nearly parallel basal bodies which are separated at their proximal ends. The four microtubular rootlets alternate between two and four members, and all are associated with a striated microtubular associated component (SMAC) that runs between the basal bodies. In addition, each half of the flagellar apparatus apparently rotates during development and loses the 180° rotational symmetry characteristic of most unicellular chlorophycean motile cells. All of these features appear necessary for efficient motion of a colony composed of numerous radially arranged cells. However, the structural details of the flagellar apparatuses of these two species differ. The distance between flagella is greater in V. rousseletii than in V. carteri. One distal striated fiber and two proximal striated fibers connect the basal bodies in V. carteri, but both types of fibers are absent from V. rousseletii. In the latter species, a striated fiber wraps around each of the basal bodies and attaches to the rootlets and the SMAC. No such fiber is present in V. carteri. Since the similarities in the flagellar apparatuses can be explained as a result of adaptation for efficient colonial motion in organisms with similar colonial morphology, the differences suggest a wider phylogenetic distance than previously believed.     

A new mutation inEscherichia coli K12,isfA, which is responsible for inhibition of SOS functions

A new mutation inEscherichia coli K12,isfA, is described, which causes inhibition of SOS functions. The mutation, discovered in a ΔpolA ⁺ mutant, is responsible for inhibition of several phenomena related to the SOS response inpolA ⁺ strains: UV- and methyl methanesulfonate-induced mutagenesis, resumption of DNA replication in UV-irradiated cells, cell filamentation, prophage induction and increase in UV sensitivity. TheisfA mutation also significantly reduces UV-induced expression of β-galactosidase fromrecA::lacZ andumuC′::lacZ fusions. The results suggest that theisfA gene product may affect RecA^* coprotease activity and may be involved in the regulation of the termination of the SOS response after completion of DNA repair. TheisfA mutation was localized at 85 min on theE. coli chromosome, and preliminary experiments suggest that it may be dominant to the wild-type allele.     

Lipid Composition and Metabolism of Volvox carteri

The membrane structural lipids of somatic cells and gonidia isolated from Volvox carteri f. nagariensis spheroids have been characterized. The principal polar lipid components of both cell types are sulfoquinovosyl diglyceride, mono- and digalactosyl diglyceride, phosphatidylglycerol, phosphatidylethanolamine, and 1(3), 2-diacylglyceryl-(3)-O-4′-(N,N,N,-trimethyl)homoserine. Light-synchronized cultures of spheroids were shown to incorporate [¹⁴C]bicarbonate, [³⁵S]sulfate, [¹⁴C]palmitic acid, and [¹⁴C]lauric acid into complex lipids. [¹⁴C]Palmitic acid was incorporated mainly into diacylglyceryltrimethylhomoserine and was not significantly modified by elongation or desaturation. In contrast, [¹⁴C]lauric acid was incorporated into a wider variety of complex lipids and was also converted into longer chain saturated and unsaturated fatty acids. Volvox is a promising system for studying the role of membranes in algal cellular differentiation.     

Ustilago maydisMating Hyphae Orient Their Growth toward Pheromone Sources

Snetselaar, K. M., Bölker, M., and Kahmann, R. 1996.Ustilago maydismating hyphae orient their growth toward pheromone sources.Fungal Genetics and Biology20,299–312. When small drops ofUstilago maydissporidia were placed 100–200 μm apart on agar surfaces and covered with paraffin oil, sporidia from one drop formed thin hyphae that grew in a zig-zag fashion toward the other drop if it contained sporidia making the appropriate pheromone. For example,a2b2mating hyphae grew towarda1b1anda1b2mating hyphae, and the filaments eventually fused tip to tip. Time-lapse photography indicated that the mating hyphae can rapidly change orientation in response to nearby compatible sporidia. When exposed to pheromone produced by cells in an adjacent drop, haploid sporidia with thea2allele began elongating before sporidia with thea1allele. Sporidia without functional pheromone genes responded to pheromone although they did not induce a response, and sporidia without pheromone receptors induced formation of mating hyphae although they did not form mating hyphae. Diploid sporidia heterozygous atbbut not ataformed straight, rigid, aerial filaments when exposed to pheromone produced by the appropriate haploid sporidia. Again, thea2a2b1b2strain formed filaments more quickly than thea1a1b1b2strain. Taken together, these results suggest that thea2pheromone diffuses less readily or is degraded more quickly than thea1pheromone.     

Genetic analysis of phosphomannomutase/phosphoglucomutase from<Emphasis Type="Italic"> Vibrio furnissii</Emphasis> and characterization of its role in virulence

The pmm gene from Vibrio furnissii, which encodes phosphomannomutase (PMM), was cloned and sequenced. The open reading frame consisted of 1,434 bp, encoding a polypeptide of 477 amino acids with a molecular mass of 53,325 Da. The predicted amino acid sequence of V. furnissii PMM showed high similarity with PMMs from other enteric bacteria, such as V. cholerae, Salmonella sp. and Escherichia coli. The PMM protein was overexpressed in E. coli as a His₆-tagged recombinant protein. The estimated apparent K_m and k_cat values of the purified recombinant protein for mannose 1-phosphate were about 60 M and 800 min^–1, respectively. To investigate the biochemical functions and the role of pmm in the virulence of V. furnissii, a pmm knock-out mutant was constructed by homologous recombination mutation. Under the various physical conditions, cell numbers of the wild-type and the mutant did not differ. Oral introduction of bacterial suspensions to a mouse model showed that the pmm-deficient mutant decreased in viability at the intestine. Microscopy of the isolated intestines from mice revealed significant damage after 3 days in intestinal mucosa infected with the wild-type as compared with the mutant. The pmm-deficient mutant caused a reduction of virulence in mice and the loss of O-antigen polysaccharide, and showed low resistance relative to the wild-type when incubated with normal human serum.     

FLAGELLAR APPARATUS STRUCTURE IS SIMILAR BUT NOT IDENTICAL IN VOLVULINA STEINII,EUDORINA ELEGANS,AND PLEODORINA ILLINOISENSIS (CHLOROPHYTA): IMPLICATIONS FOR THE “VOLVOCINE EVOLUTIONARY LINEAGE”1

The colonial and multicellular members of the Volvocales can be arranged in order of increasing size and complexity as the “volvocine series.” This series is often assumed to reflect an evolutionary progression. The flagellar apparatuses of previously examined algae are not consistent with a simple lineage. The flagellar apparatuses of Astrephomene gubernaculifera Pocock, Gonium pectorale Müller, Platydorina caudata Kofoid, Volvox rousseletii G. S. West, and V. carteri f. weismannia (Powers) Iyengar differ from one another, and there is no apparent progression inflagellar apparatus features from the simple to complex colonial forms. We examined the flagellar apparatuses of Volvulina steinii Playfair, Eudorina elegans Ehr., and Pleodorina illinoisensis Kofoid and found them to be similar to one another. The basal bodies are connected by a distal fiber that is offset to the anti side of the cell. Two microtubular rootlets originate on the inside of the basal bodies and extend toward the syn side. The other two rootlets are oriented perpendicular to the first two and are anti-parallel to each other. A coarsely striated component underlies the four-membered rootlets and extends to the basal bodies. A proximal fiber complex connects the two basal bodies. This complex consists of a branched striated component on the cis side of each basal body. One part extends toward the anti side of the cell, while the other extends into a fibrous component that runs between basal bodies. An additional structure extends in the anti direction from the trans side of each basal body. A fibrous component extends past one basal body in all four species. This component goes past the trans basal body in Volvulina steinii and the cis basal body in E. elegans and P. illinoisensis. The flagellar apparatuses of these organisms are similar to those of G. pectorale and Volvox carteri but different from the other colonial volvocalean algae examined. The algae examined in this study plus G. pectorale and V. carteri probably share a common evolutionary history that postdates the transition from the unicellular to colonial habit. Such a shared evolutionary history is a requirement of the volvocine hypothesis. However, we have not observed progressive changes in the flagellar apparatus correlated with increasing cell number, differentiation, and sexual specialization. Thus, it is possible, but not certain, that G. pectorale, Volvulina steinii, E. elegans, P. illinoisensis, and Volvox carteri may form part of a volvocine lineage.     

Improved production and stability ofE. coli recombinants expressing transketolase for large scale biotransformation

Summary TheE.coli tkt gene has been subcloned into high copy number vectors. In fed batch fermentations up to 4gL^–1 of soluble intracellular transketolase was produced representing 43% of the total cell protein. Increased plasmid stability during fed-batch fermentations was obtained by using kanamycin resistant pBGS vectors rather than the ampicillin resistant pUC vectors. Plasmid stability was maintained throughout growth in a complex medium without any selective pressure by incorporating thecer region fromColE1 into the expression construct.     

Effect of juglone on growthin vitro ofFrankia isolates and nodulation ofAlnus glutinosa in soil

Summary In vitro growth (total protein content) of 5Frankia isolates was significantly inhibited at 10^–4 M juglone (5-hydroxy-1, 4-napthoquinone) concentration, but the degree of inhibition varied with theFrankia isolate. Isolates fromAlnus crispa [Alnus viridis ssp.crispa (Ait.) Turril] were most tolerant of 10^–4 M juglone relative to controls, while an isolate fromPurshia tridentata (Pursh.) D.C. was most inhibited, displaying a dramatic decrease in growth and greatly altered morphology.Nodulation of black alder [Alnus glutinosa L. (Gaertn.)] in an amended prairie soil inoculated with aFrankia isolate from red alder (Alnus rubra Bong.) was significantly decreased by the addition of aqueous suspensions of 10^–3 M and 10^–4 M juglone. This decrease was partially independent of decreased plant growth. The addition of an equal volume of sand to the soil mixture further decreased nodulation of black alder.Frankia inoculation of the soil mixtures significantly increased the total number of nodules formed per seedling, and the degree of differences in seedling nodulation owing to juglone and soil treatments.     

Characterization of the superoxide dismutase of the denitrifying bacterium,Bacillus halodenitrificans

Summary Bacillus halodenitrificans produced a dimeric, manganese-containing superoxide dismutase constitutively when grown either aerobically or as a denitrifier. The molecular mass of the enzyme was determined by sedimentation equilibrium to be 41.4±3 kDa with each subunit estimated at 26 kDa. Plasma emission spectroscopy indicated the presence of 1.22 mol manganese atoms/mol holoenzyme. The electronic absorption spectrum displayed a broad band centered at approximately 474 nm (=560 mM^–1 · cm^–1) and a shoulder at 595 nm. In the ultraviolet range, the spectrum exhibited split maxima at 278 nm and 283 nm and a shoulder at 291 nm, thus resembling the spectra of superoxide dismutase fromBacillus subtilis andEscherichia coli. The amino acid composition of theB. halodenitrificans enzyme differed slightly quantitatively but little qualitatively from counterpart enzymes from other sources. Like the superoxide dismutases ofMycobacterium lepraemurium and human mitochondria, theB. halodenitrificans enzyme exhibited several cysteine residues. As expected from the capacity superoxide dismutase exhibits for protecting NO as neutrophil cytotoxicity factor, theB. halodenitrificans superoxide dismutase did not interfere with accumulation of NO produced by the organism's nitrite reductase.